The low molecular weight NADH dehydrogenase which can be solubilized from the mitochondrial NADH-ubiquinone oxidoreductase complex with chaotropic agents consists of three subunits in equimolar ratio [Galante, Y. M., & Hatefi, Y. (1979) Arch. Biochem. Biophys. 192, 559]. The largest subunit (subunit I) can be completely separated from the other two (subunits II + III) by treatment with sodium trichloroacetate and ammonium sulfate fractionation. Both the subunit I and subunit II + III fractions contain iron and acid-labile sulfur. From visible and EPR spectroscopy and the iron and acid-labile sulfide content, we propose that the subunit II + III fraction contains a binuclear cluster. The cluster structure present in subunit I is as yet unclear. On separation of the subunits of NADH dehydrogenase, the FMN is lost.